How the cochleae of humans and other mammals achieve their remarkable sensitivity, frequency selectivity, and enormous dynamic range has been the central question in auditory neurobiology. Two competing mechanisms have been proposed: the mammalian-specific prestin-based outer hair cell (OHC) electromotility and the ubiquitous stereociliary motility. In the previous funding period, we demonstrated that: (1) prestin-based OHC electromotility is necessary for cochlear amplification;(2) prestin plays a novel role in frequency tuning of cochlear passive mechanical responses and their transmission to neural excitation;(3) prestin based OHC electromotility does not appear to adjust the operating point of stereociliary motility;and (4) Glut5, a previously hypothesized OHC motor protein, is undetectable in OHCs and does not contribute to cochlear amplification. Based on these and other advances, we propose a unified amplificatory mechanism that stipulates stereociliary motility for tuning and electromotility for power. However, it remains controversial whether prestin plays both active and passive mechanical roles and whether prestin-based electromotility performs the necessary cycle-by-cycle feedback. Furthermore, it is still unclear how prestin drives coordinated changes in the lateral plasma membrane and underlying cytoskeletal structure for OHC electromotility. To further elucidate prestin's roles in OHC electromotility and cochlear amplification, we will pursue the following Specific Aims: 1) Determine how prestin-based OHC electromotility generates cochlear amplification. 2). Determine the distribution, trafficking, and membrane mobility of prestin in OHCs. Recently, two putative mutations in the human prestin gene have been reported to cause deafness. Hearing loss induced by large doses of sodium salicylate (aspirin) has been attributed to a reduction in prestin-based OHC electromotility. Moreover, prestin is likely the common effector of hearing loss in some patients with high-frequency hearing loss. Our studies will contribute greatly to our understanding of cochlear amplification and the pathophysiology of deafness caused by a variety of genetic and environmental factors. PUBLIC HEALTH RELEVANCE: An estimated 28 million people in the United States are deaf or hard of hearing. Approximately 1.5 million individuals aged 3 years or older are deaf in both ears and 2 to 3 per 1,000 live births suffer congenital hearing loss. More than 40 million persons in the United States suffer various levels of noise induced hearing loss. Nearly half of people over 65 years of age develop age-related hearing loss. Despite the significant progress in our understanding of these hearing disorders, very little is known about the disease causes and about the normal hearing processes in adults. Here we propose to study prestin, a motor protein in the inner ear that is crucial for our hearing sensitivity and frequency selectivity. Recently, two putative mutations in the human prestin gene have been reported to cause deafness. Hearing loss induced by large doses of sodium salicylate (aspirin) has been attributed to prestin-mediated hearing dysfunction. Moreover, prestin is likely the common effector of hearing loss in some patients with high-frequency hearing loss. Our studies will contribute greatly to our understanding of cochlear physiology and the pathophysiology of deafness caused by a variety of genetic and environmental factors.